Identification and localization of myosin phosphatase in human platelets.
Type 1 (PP1) and type 2A (PP2A) phosphatase activity was measured in three subcellular fractions of human platelets. About 80% of the activity was in the high-speed supernatant. Western blots showed that the catalytic subunit of PP1 (PP1c), including alpha- and delta-isoforms, was present in each fraction, but the level of the catalytic subunit of PP2A was very low in the low-speed pellet (cytoskeletal fraction). Various antibodies detected a subunit similar to the 130 kDa subunit (M130) of myosin phosphatase (MP) of smooth muscle in the low- and the high-speed pellets of human platelets. PP1c and associated proteins were isolated by microcystin-Sepharose. Many proteins were separated from each fraction, including myosin, actin and PP1c. M130 was separated only from the low-speed and the high-speed pellets. Kinase activities were detected in the unbound fractions, and fractions from the low- and high-speed pellets phosphorylated M130 and myosin respectively. Treatment of platelets with calyculin A increased the phosphorylation level of many proteins, including myosin heavy- and light-chains, and caused association of cytoskeletal proteins with the low-speed pellet. No marked change in the distribution of PP1c and M130 was detected. These results suggest that the MP in human platelets is composed of PP1c plus a subunit similar to M130 of the smooth muscle phosphatase.
Murányi
A
A
Department of Medical Chemistry, University Medical School of Debrecen, H-4026 Debrecen, Bem tér 18/B, Hungary.
Erdodi
F
F
Ito
M
M
Gergely
P
P
Hartshorne
D J
DJ
D000328:Adult; D001792:Blood Platelets / Q000201:enzymology*; D015153:Blotting, Western; D003599:Cytoskeleton / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D018994:Myosin Light Chains / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases / Q000032:analysis*; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D054645:Protein Phosphatase 1; D013347:Subcellular Fractions / Q000201:enzymology
D046911:Macromolecular Substances; D018994:Myosin Light Chains; D010750:Phosphoproteins; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D043263:Myosin-Light-Chain Phosphatase